Bovine profilin isoforms bind both the lipid phosphatidylinositol
4,5-bisphosphate (PIP2) (the target of Phospholipase C - see Protein kinase C for more information) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein
(VASP) (see Enabled) and cyclase-associated protein (CAP). Compared with
profilin II, profilin I has a higher affinity for PIP2. However, proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II
dimerizes on binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a
significant conformational change in this protein upon binding of the peptide.
PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin
II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either
profilin isoform, profilin II was identified as the preferred ligand for VASP. The
complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an
opportunity to direct actin assembly at different subcellular localizations through the same or different
signal transduction pathways (Lambrechts, 1997).

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI
4,5-P2) and regulate the concentration of monomeric actin. Profilin and gelsolin
stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a
concentration-dependent manner. This effect is specific to profilin and gelsolin because other
cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase
activity, PI 3-kinase also carries out protein kinase activity: it phosphorylates proteins (p85 subunit of PI
3-kinase). However, the protein kinase activity of PI 3-kinase is not affected in the presence of
profilin. Profilin may also affect PI 3-kinase
activity by its direct association to the enzyme.
However, PI 3-kinase does not affect the actin-sequestering ability of profilin indicating that actin and p85 do not share a common binding site on profilin.
These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated
phosphoinositides, which in turn may regulate actin polymerization (Singh, 1996).

Although profilin's interactions with its three known
ligands (poly-L-proline (PLP), phosphatidylinositol 4,5-bisphosphate (PIP2), and actin monomers) have
been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters
of PIP2, profilin is able to inhibit the hydrolysis of PIP2 by phospholipase C gamma 1 (PLC gamma 1).
This ability is the result of profilin's affinity for PIP2, but the specific residues of profilin's amino acid
sequence involved in the binding of PIP2 are not known. The following mutants of human
profilin were used (named according to the wild-type amino acid altered, its position, and the amino acid
substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D,
and K125Q. With the exception of L10R, all of the mutants were successfully expressed in
Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q
demonstrate moderately less stringent binding to PLP, indicating that most of the mutations do not
induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the
hydrolysis of PIP2 by PLC gamma 1, most of the mutants are indistinguishable from wild-type
profilin. Exceptions include D8A, which demonstrates increased inhibition of PLC gamma 1, and
R88L, which demonstrates decreased inhibition of PLC gamma 1. To assess the importance of the
region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to
non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC
gamma 1. Only the decapeptide that matches the peptide stretch centered around residue
88 is able to inhibit PLC gamma 1 activity substantially and is able to do so at nearly wild-type
profilin levels. Taken together with the finding that mutating residue 88 results in decreased inhibition
of PLC gamma 1 activity, these data provide strong evidence that this region of human profilin
represents an important binding site for PIP2 (Sohn, 1995).

Depolymerization of actin filaments by profilin: effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microM). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. The effects were examined of higher concentrations of profilin (10-100 microM) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, 40 microM), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin has no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration is independent of monomer sequestration. Furthermore, it was found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments has not been appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization (Bubb, 2003).

Profilin response to Rho family members and interaction with Diaphanous homologs